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Sartorius is investing approximately €270 million (US$305 million) through the end of 2024 to expand its activities in South Korea, the company announced on Nov. 25, 2021. Using the investment funds, the company plans to launch cell culture media production in Songdo, Incheon, South Korea’s biopharma hub, in addition to assembling sterile systems for efficient and flexible biopharmaceutical manufacturing, among other operations.

Construction of new facilities at the 25,000-m2 (270,000-ft2) site will include an application center with laboratories as well as several buildings for manufacturing and logistics. Construction is slated to begin in the coming year with production operations scheduled to start by the end of 2024. The expansion will create approximately 750 new jobs. Sartorius’ existing activities for service as well as for marketing and sales will be relocated to the new site.

“The development of the biopharmaceutical industry in Asia and, in particular, South Korea, has entered a new phase characterized by a high innovation and investment pace on the part of our customers. With our significant investments in Songdo, we will be increasing capacities, moving production operations closer to our customers in the region and substantially accelerating our delivery speed,” said Joachim Kreuzburg, executive board chairman and CEO, Sartorius, in a company press release.

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Sartorius will invest approximately €270 million (US$305 million) to expand its biopharmaceutical manufacturing activities in South Korea’s biopharm hub.

Sartorius Launches Expansion in South Korea with Mullti-Million Dollar Investment

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.

Defining cell and virus quality is the heart of successful cell and gene therapy production. Process analytical technology (PAT) that enables real-time monitoring and feedback control of manufacturing processes has the potential to increase productivity and product quality. Some solutions are currently available and being applied in the industry, but they do have limitations.

Greater process and product understanding and advances in fundamental technologies will be required before widespread application of PAT occurs for cell- and gene-therapy manufacturing. The excitement around these novel medicines will likely, however, help drive that needed innovation.

Numerous benefits of real-time monitoring and feedback control

Real-time monitoring and feedback control for cell- and gene-therapy manufacturing would allow real-time decision-making, reduce processing bottlenecks, and enhance process reproducibility and batch-to-batch comparability, according to Inbar Friedrich Ben-Nun, associate director of cell therapy R&D at Lonza. Making time-sensitive information available at a point where forward manufacturing decisions can still be made increases the value of process data, adds Richard Harrison, collaborations manager with Cell and Gene Therapy Catapult.

Reduction of manual process interventions would also result in decreased batch variability and/or failure, and lower the cost of manufacturing by reducing the use of various offline equipment and labor for analysis, Friedrich Ben-Nun says. Reductions in costs could also be realized through intensification of processes into smaller footprints and a reduction in number of operators required to run processes, while earlier alerts when failure does occur would help minimize lost product and the allocation of resources to failed batches, observes Harrison.

In addition to the development and implementation of robust and consistent manufacturing processes, real-time monitoring and feedback control enable a more flexible, tunable approach to manufacturing rather than a “one-size-fits-all” strategy, comments Pratik Jaluria, global head of technical development for cell therapies at Novartis. That would be particularly beneficial with respect to affording higher yields and accounting for donor-to-donor variability with autologous therapies, John Churchwell, senior scientist, Technology and Process Innovation at Cell and Gene Therapy Catapult, remarks.

Deeper insights into key inputs/outputs that could be predictive of clinical efficacy and safety would also be enabled by real-time monitoring and feedback control, according to Jaluria. Reductions in analysis times, meanwhile, would shorten the overall product release timeline, says Francesca Rossetti, analytical methods development manager with AGC Biologics’ Milan Cell & Gene Therapy Center of Excellence. “Real-time release of products,” concludes Churchwell, “would save time and money and get treatments to patients faster.”

The application of a quality-by-design (QbD) approach and PAT for real-time monitoring and feedback control for advanced therapy
medicinal product (ATMP)-manufacturing first requires the identification of relevant critical process parameters (CPPs), which can be challenging. “While many critical quality attributes (CQAs) are intuitive, such as quality, purity, and potency for cell therapies and additionally titer, infectivity, and empty/full capsid ratios for viral vectors, the process parameters that relate to these attributes are less obvious,” Churchwell explains.

“An effective approach is to investigate these correlations using a biomarker identification strategy that involves applying bioinformatic and systems-biology tools such as pathway analysis and canonical correlation analysis,” Churchwell believes.

Targeting upstream control for cell therapies

Cell quantity and quality are critical for the efficacy of cell-therapy applications and patient safety. Current cell-therapy manufacturing processes are, however, relatively fragmented, according to Brian Hassell, founder and CTO of Nirrin Technologies. “The goal for future PAT solutions, therefore, will be to connect all of the pieces of the cell therapy process,” he states.

Upstream cell-therapy processes, says Friedrich Ben-Nun, are usually continuous and involve long culture periods that allow for cell activation, cell expansion, and cell maturation or differentiation. Cell density can impact many aspects of culture processes, from gas exchange to the frequency of media replacement. Changes in the process, meanwhile, can affect cell viability and performance.

“For these reasons, it would be ideal during upstream processes to monitor cell density, cell viability, nutrients/metabolites in the culture media, growth factors in the media, cell performance (i.e., cytokine secretion), cell identity (via cell-surface markers or genetic markers), and bioburden,” Friedrich Ben-Nun comments.

Downstream processing of cell therapies, however, is generally different than that for traditional biologics. Cells are not commonly purified through a traditional chromatography column, though magnetic columns can be used with soluble components being added first to the cells, Jaluria explains.

“Considering the short term of the downstream process for cell therapies, it might be redundant or plethoric to apply PAT solutions, especially if they have not been adopted by regulatory authorities as substitutes for traditional cell-based assays. Nevertheless, this situation can lead to at-risk processing until the desired parameter can be measured,” Friedrich Ben-Nun says.

Jaluria adds that because downstream processing for cell therapies is really more about cell washing and buffer exchange, fill/finish technology equipped with sensors enabling real-time monitoring of specific quality attributes would be highly sought after.

Ideally, Jaluria observes, the impact of each unit operation on cell recovery including desired populations, cellular metabolic states, and other cellular properties would be evaluated. “In general,” Hassell concludes, “we need to be able to turn the knobs on transduction and expansion culture parameters while observing quantitative readouts for cellular identity, quantity and quality/function and have similar capabilities for downstream to ensure that final purification, formulation and storage do not affect any of the aforementioned attributes before administration of the product to the patient.”

Additional virus-related monitoring needed for viral therapies

While for viral vectors the cells are not the therapeutic product, many of the same process parameters need to be monitored during upstream processing (e.g., transfection or infection of cells). Additional upstream CQAs that should be monitored include viral particle production and packaging (full/partial/empty capsids), according to Friedrich Ben-Nun. Rossetti agrees that real-time monitoring would also be useful in viral particles testing to determine the best timing for vector harvest.

During downstream processing, Friedrich Ben-Nun says there is a need to continuously monitor the process, verify the viral product concentration, test for bioburden, identify empty vs. full capsids, and measure aggregates and other process-related impurities. “Particle aggregation,” stresses Rossetti, “is a very important parameter that should be monitored in real-time in order to reduce the risk of a low filtration yield.”

In general, Jaluria believes that PAT can aid in reducing the batch-to-batch variability commonly observed in current viral-vector manufacturing processes, which can impact a host of outputs including yield and various characteristics associated with release testing. “The implementation of PAT during all stages of production can improve process efficiency and increase product quality and safety,” Friedrich Ben-Nun confirms.

Application of PAT for cell and gene therapy production in its infancy

At present, only a few PAT solutions are being implemented for cell- and gene-therapy manufacturing. Many are the same as those used for cell-culture processes for traditional biologics, including sensors for monitoring dissolved oxygen, pH, temperature, and other basic process parameters. Most analyses for CQAs, however, are still performed offline, limiting their value for enhancing process control, Hassell states.

Enzymatic patch sensors for glucose/pH and basic capacitance-based pH probes are probably the most widely used PAT solutions today, observes Harrison. For more complex analytical monitoring, he notes that Raman spectroscopy represents the most mature of the complex spectroscopic PAT tools. Overall, though, Churchwell agrees that in the public domain there is limited application of advanced PAT in upstream ATMP manufacture and PAT in cell- and gene-therapy manufacturing is in its infancy.

The application of advanced PAT techniques such as Raman and 2D fluorescence spectroscopy have been shown, though, to enable real-time monitoring of a large number of additional parameters, Churchwell states. Examples include glucose, lactate, glutamine, glutamate, ammonia, total and viable cell density (TCD/VCD), product titer, cofactors, vitamins, and amino acids. Capacitance/dielectric spectroscopy also allows the direct monitoring of viable biomass, he says.

Specifically for some viral vectors, Jaluria adds that in-process sizing (i.e., cell size changes) and visualization are commonly used for upstream monitoring, with technologies that evaluate particle formation (charge-detection mass spectrometry, cryoelectron microscopy, and reverse-phase high performance liquid chromatography) increasingly being applied to intermediates.

During downstream purification, Friedrich Ben-Nun points to in-line pressure measurement during depth filtration as a PAT solution useful for indicating clogging for both cell therapies and viral vectors. She also notes that inline pressure and conductivity measurements are often collected during tangential filtration used for the harvest and formulation steps, while inline pressure, pH, conductivity, and UV absorbance monitoring are implemented during column chromatography for viral vectors.

Multi-angle light scattering (MALS), notes Churchwell, allows determination of the molecular weight of adeno-associated virus (AAV) vectors and quantification of the empty/full capsid ratio in downstream purification.

There are many reasons why PAT has not yet been widely implemented for cell- and gene-therapy manufacturing. At a fundamental level, there remains limited access to suitable technologies due to the need for advances in analytical technologies. Expertise in PAT is also needed, as in many instances existing processes/workflows need to be significantly modified before PAT can be implemented, according to Jaluria.

“The most widely in-process measurements only provide very basic process information. While the more complex spectroscopic PAT solutions such as Raman and other spectral techniques can give increasingly rich datasets, they are harder to robustly implement into a process and require dedicated expertise and experimental discovery runs in order to achieve performance at the required level,” Churchwell elucidates. “All advanced PATs have strengths and weaknesses; there is often a trade-off between sensitivity and chemical specificity,” he adds.

Robustness is also essential, stresses Hassell, because every company runs processes differently. Achieving a sufficient dynamic range for PAT sensors is an incredibly difficult problem to solve, however, primarily because processes can vary by two to three orders of magnitude, and manipulation of samples (concentration, dilution) introduces error. “For effective PAT, sensors need to be able to adapt to dynamic range and work across all scenarios,” he insists.

The complexity of cell- and gene-therapy manufacturing processes is also an important factor, according to Friedrich Ben-Nun. Measuring key factors in culture media, such as small molecules and growth factors, is generally not possible with most current PAT solutions. The complexity of the raw materials used in these processes can also hinder adoption of current PAT techniques, she says. In addition, the need for a high level of product understanding necessitates multiple experiments, with any process change requiring repetition of that work. The result, at least initially, can be manufacturing delays and/or deviations, which can put the process and product at risk.

More product understanding, better sampling, and challenges

The need to gain better product and process understanding—by running many experiments to enable linkage of product CQAs to CPPs—is, according to Friedrich Ben-Nun, an important challenge facing the implementation of PAT for cell- and gene-therapy manufacturing. Another is the large number of unit operations involved in some manufacturing processes, which means large numbers of CPPs that must be measured and higher capital investment associated with PAT implementation.

In addition, while regulatory agencies are encouraging the use of PAT for traditional biologic processing and would likely be accepted or even encourage PAT for cell-
and gene-therapy manufacturing, Friedrich Ben-Nun observes that it is not clear if PAT solutions could replace current offline bio-assays to determine, for instance, cell performance and bioburden levels.

There are fundamental technical challenges as well. “Much of the available tools were not explicitly designed for cell- and gene-therapy applications and may need re-designing or qualification prior to use,” explains Churchwell. “We are therefore likely to see a number of new devices tailored for this market emerging over the coming years that allow the connection of relevant advanced PAT solutions,” he adds.

For cell therapies, Jaluria points to the need to get a meaningful measurement of cellular properties quickly and in such a way that the material is not changing during sample preparation and manipulation so that the result is representative of the bulk that continues to be processed. “Reliable inline sensors that can provide release attributes in real time have yet to be developed,” he says.

For viral vectors, there are challenges around the small particle size of these materials, some of which can only be quantified using molecular methods, such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) techniques, according to Jaluria.

Sample size is also an issue. PAT methods must be developed that are highly sensitive and require only very small volumes given that process intermediates are precious  materials. Instrument capabilities, Hassell remarks, need to accommodate both small samples and wide dynamic ranges. 

In that vein, instruments must also be able to work with minimal
calibration and still give accurate measurements, particularly when PAT is being applied to autologous therapies. “All patients are different, and there is tremendous variability in processes and products, so robustness is essential across unknown scenarios,” underscores Hassell. Standardization of integration across scales and
platforms and wide acceptance of reliable scale-down models for developing and implementing PAT solutions are needed.

The lack of organizational awareness of the QbD methodology as applied to ATMPs and incorporating PAT to enable digitized manufacturing is another great challenge, according to Churchwell. “There is limited understanding of automation, physical techniques, and data-analysis/statistical-modeling methodologies. Implementing PAT solutions requires an integrated approach involving biomarker/CQA identification, PAT sensor application and the concomitant application of design-of-experiment approaches,” he explains.

Rossetti adds that the complex and time-consuming nature of most assays used for the characterization of ATMPs is a significant hurdle to the development of effective PAT solutions. “Many of these assays cannot easily be performed in a manner that generates results in real-time,” she explains. At AGC Biologics, to help reduce these challenges, the company developed a custom analytical platform. Optimization of the number and volume of samples required for quality control allows for faster turnaround times and reduced product consumption.

An industry-wide and collaborative effort to advance solutions

The interdisciplinary nature of cell and gene therapies has led to collaboration on research and development efforts spanning the fields of physics, chemistry, cell biology, engineering, computer science, and many others.

Greater dialogue between health authorities and drug developers around the numerous challenges facing cell and gene therapy manufacturing could also lay the foundation for how new technologies can be assessed and implemented, Jaluria believes. “One potential outcome of this interaction could be the preparation of general guidelines in which national or international agencies provide avenues and best practices around novel analytical technologies, progressing the field,” he says.

Industry-wide collaborations such as the National Institute for Innovation in Manufacturing Biopharmaceuticals, BioPhorum Cell & Gene Therapy, and Cell and Gene Therapy Catapult are, meanwhile, allowing companies to partner in advancing solutions.

As one specific example, Cell and Gene Therapy Catapult formed the PAT consortium to accelerate technology development and potentially lower the cost of cell-and gene-therapy manufacturing. This consortium will build industry awareness and leverage relevant skillsets to enable more rapid uptake of PAT technologies and digitized automation strategies and sensor integration. The consortium comprises more than 20 organizations ranging from pharmaceutical companies and technology providers to therapy developers and charities.

One goal of the PAT consortium is to act as a catalyst in accelerating the development of the necessary knowledge and understanding at reduced cost and investment risk to each organization.

“By reducing the barrier to entry for these technologies, we hope that more companies are able to take advantage of these techniques,” notes Harrison.The PAT consortium is the first large-scale dissemination activity in this field, but other “exciting future collaborations” can be expected. “With these efforts and the efforts of those across the industry, we expect to see an increase in the number of vendors supplying cell- and gene-therapy-specific instrumentation and a reduction in the barrier to entry for all cell- and gene-therapy companies to take advantage of these techniques in their processes,” Harrison ventures.

Companies such as Lonza and Novartis are constantly evaluating potential new technologies that may overcome the challenges facing the industrialization of cell and gene therapies. “PAT is an active area of research, and a number of companies are looking to introduce their solutions in the next few years that could address many of the current bottlenecks,” Jaluria observes.

Rossetti points to the use of microfluidic systems to perform automated protein analyses (ELISA, Western Blot) as solutions that would allow operators to analyze more samples in the same test run, reducing time and cost. She cautions, though, that while these new and automated technologies show promising features, their development must be further advanced to ensure compliance with GMP regulatory requirements.

Nirrin Technologies is focused on developing PAT solutions based on near infrared (NIR) technology that address specific industry issues. “We use NIR optical sensors combined with machine learning to enable the next generation of upstream and downstream sensor solutions,” Hassell explains. He notes that the company is focused on the largest unmet needs, including easy-to-use, robust, accurate measurements for glucose, VCD/TCD and titer, with an emphasis on control system integration using the DeltaV Distributed Control System from Emerson.

Ultimately, the goal is to have real-time monitoring methods that can be implemented both upstream and downstream that require minimal sample volumes, enable comprehensive characterization of nucleic acids (RNA, DNA) and protein targets, and can be paired with control software for unit operations that allows for easy data analysis.

“At the deepest level,” states Hassell, “we want sufficient -omics content for understanding cell function, identity, and quality. For example, proteomics and transcriptomics combined with cytokine/chemokine analysis. Combined with data on more macroscopic properties such as cell number quantitation, viral titer, and full-versus-empty status for viral vectors, this type of solution would support the ultimate manufacturing platform.”

At Cell and Gene Therapy Catapult, the goal is to identify novel techniques and/or techniques that are new to the field that can address in-process measurement challenges in the industry. “In this respect, we are agnostic as to the specific methods employed, provided they help us measure identified CPPs and CQAs,” Churchwell comments.

Specific PAT solutions that Friedrich Ben-Nun would like to see realized include a rapid real-time method to monitor growth factors during cell production; a real-time method to identify cell-type impurities such as immature or undifferentiated cells during manufacturing; a real-time method for detecting cell characteristic changes (cell shape, nucleus-to-cytoplasm ratio, hormone secretion, etc.) as an indicator of process success; a real-time method for monitoring virus particle number, aggregation, and genomic content; biomarker-based methods for correlating or predicting product potency; a rapid real-time method to identify impurities during viral-vector purification; and incorporation of automated sample systems with targeted analytical solutions.

For vector production processes, Rossetti says that rapid ELISA assay testing with microfluidic technology for quantification of the p24 capsid protein could help to immediately understand the production yield and decide if a team should proceed with downstream processing. She also notes that real-time analysis of process-related contaminates during downstream processing would also be valuable. For cell therapies, Rosetti would also appreciate a PAT system that can analyze not only viability, but immunophenotype as well, to better characterize cell subpopulations and evaluate the level of differentiation for products that are ultimately being infused into patients.

As the field continues to mature, cell- and gene-therapy manufacturing are perfectly positioned to benefit from advances in PAT technology developed for mammalian manufacturing of traditional biologics. “Fine-tuning and optimization of the tools developed for mammalian manufacturing of biologics will be critical to enable the adoption of PAT for cell- and gene-therapy manufacturing,” Friedrich Ben-Nun says. She adds, though, that new PAT solutions with specific applications to cell- and gene-therapy manufacturing will need to be developed in order to support the variable process pipeline in cell and gene therapy.

Jaluria agrees that there are great opportunities in the space of inline and nondestructive analytics for use in the production of cell and gene therapies. Hassell goes even further: “The timing is ideal for PAT in cell and gene therapy. It will start to succeed because of the immense amount of research and excitement surrounding ATMPs such as cell and gene therapies, which is driving the development of novel, next-generation biological sensors.”

Cynthia A. Challener, PhD is contributing editor to 


Vol. 34, No. 12
December 2021
Pages: 10–14

When referring to this article, please cite it as C. Challener, “Process Analytical Technologies for Manufacturing Cell and Gene Therapies,” 

Timing is everything, and it might be ideal for acceleration of real-time monitoring solutions. 

Process Analytical Technologies for Manufacturing Cell and Gene Therapies

Raymond P. Goodrich, PhD, is the founder of PhotonPharma, executive director of the infectious disease research center at Colorado State University, and professor for the department of microbiology, immunology, and pathology at Colorado State University.

Amanda Guth, DVM, PhD, is the chief scientific officer for PhotonPharma.

Jon Weston is the president and CEO of PhotonPharma.

Terry Opgenorth, PhD, is the executive in residence life sciences for CSU Ventures.

Gary Gordon, MD, PhD, is the president of Global Coalition for Adaptive Research.

Traditional approaches to treating patients with cancer are based primarily on maximizing the destruction of tumor cells while minimizing the damage to healthy cells. The ability of chemical agents, radiation, and surgical procedures to destroy or remove tumor cells while preserving the functionality of healthy tissue is the basis of every form of cancer therapy. The result of this quest has yielded therapeutic formulations targeting unique metabolic, genomic, and phenotypic characteristics of cancer cells for the treatment of patients with cancer.

Advances in our understanding of immunology and the body’s ability to defend itself against aberrant cells and tissue have afforded new tools in our search for better treatments and cures. Most of these approaches rely on the body’s inherent ability to distinguish between cancerous cells and healthy tissue and to respond through both the production of antibodies (humoral immunity) and the generation of cell-based immune responses. New formulations of autologous therapies for cancer are produced by culturing and expanding specific immune cell populations, specifically T cells (T lymphocyte), 

 and training them for seek-and-destroy missions when re-infused. These therapies have produced life-saving outcomes in patients, such as those suffering from B-cell lymphomas. Nonetheless, these methods have limitations such as non-specific targeting (e.g., destroying all B cells and not just the cancerous ones), cost of production, and complexities related to 

The complexity of steps involved in the production of targeted T cells require 

 expansion and manipulation of the immune cells, which in turn requires costly reagents to produce enough cells for administration. These manufacturing steps drive up cost, introduce logistical issues, and reduce the ability to treat the diverse nature of most tumors. For autologous therapies for cancer, new therapeutic formulations not only use the patient’s own immune cells but also their own tumor cells to generate therapeutic preparations for treating their specific cancer. This approach may address many of the current limitations of cell-based immunotherapy.

Patient-specific immune therapy limitations

The development of methods to manipulate immune cells from a patient 

 has greatly increased the utility and benefit of cellular therapy and autologous, patient-specific immune therapies. The most recent example of Chimeric Antigen Receptor (CAR) T-cell therapy targeting B-cell lymphoma is perhaps the most specific illustration of how personalized cell therapy approaches can be effectively implemented with great benefit to patients (1). By developing methods to target CD19 antigen, 

, T cells are generated with the ability to seek and destroy all cells expressing CD19 antigen. Both tumor cells and healthy B cells with this antigen are eliminated. The resulting reset of the immune system through triggering mechanisms that silence these altered T-cell clones can lead to the cure of the lymphoma that the patient has on board.

Unfortunately, a reset such as this is not possible with other cell types. While it is feasible for an individual to survive for a period without B cells, it is not possible for them to survive if all lung, colon, liver, and/or other solid organ tissue were similarly targeted for elimination. The limitation of T-cell therapies of this type are thus dependent on identifying antigens that are specific to the tumor (neoantigens) but not present on the healthy cells. The presence of such specific antigens allows the immune system to distinguish between healthy and cancerous and thus eliminate the tumor more specifically. Unfortunately, targeting against a specific neoantigen may prove insufficient since tumors may contain a plethora of these tumor-specific antigens, which are not expressed across every cell in an individual’s tumor. This issue can lead to the escape of some tumor cells, resulting in tumor regrowth if the targeting is too specific. In recent years, multiple new cell therapies have entered clinical trials and are reporting improved efficacy over the standard of care (2). These therapies are using genomic analysis and artificial intelligence to identify or predict neoantigens on individual patient tumors. Researchers then construct peptide vaccines or potentiate harvested patient immune cells to the neoantigens for reinfusion.

T-cell culturing and dendritic cell manipulation 

 also require a complex logistical framework for manufacture. First, immune cells must be harvested from the patient in sufficient quantities to allow subsequent culturing and expansion. These processes often take weeks of 

 manipulation in cell bioreactors using reagents and culture conditions that promote cell expansion of specific cell types to the exclusion of others. An insufficient yield of cells at the end of culture could mean that the patient is unable to subsequently receive an effective therapeutic dose of the altered cells. The 

 manipulation of the product required also introduces challenges in maintaining sterility and covering high costs associated with the reagents and procedures needed to drive cell differentiation in culture along the desired pathways. The average cost of CAR-T therapy exceeded $400,000 per treatment in the initial introduction, and reimbursement today remains at $240,000 in most settings (3). This limits the number of patients for whom such treatment is accessible.

An alternative approach: CAR-T therapy in reverse

 manipulation of the immune cells is to modulate immune responses 

 by presenting the tumor antigens to the immune cells in an environment separate from the tumor microenvironment where immune suppressors may be expressed by the tumor cells, thus reducing immune response at the local site of the tumor. Such an approach would allow the type of cell modification that CAR-T therapy achieves 

 through the presentation of tumor antigen to the immune system that the patient has on board.

Several methods using specific tumor antigens or whole tumor cells, which have been inactivated before administration, have been utilized previously with moderate success (4). 

Single antigen approaches, however, face the same challenges that CAR-T cell therapy faces. Elimination of specific clones of a tumor may not lead to complete clearance of all tumor cells in a patient. The use of whole, inactivated cells requires that the cells be rendered incapable of replication post-infusion to prevent the regrowth of tumors in the patient. To assure complete inactivation, common methods employ chemical agents such as formalin, ultraviolet (UV) light, or gamma irradiation of sufficient energy and dose to destroy cell replication processes. The harsh and non-specific nature of the chemistry that these methods invoke results in cells that are not only inactivated but also rendered altered from the native antigen state that these tumor cells express. As a result, these methods often require large amounts of tumor material to induce a strong immune response, effectively increasing the dose to levels at which sufficient amounts of the native tumor antigen are still capable of generating immune reaction to those antigens.

Currently, we are exploring the ability of a method initially developed to treat blood products for the prevention of transfusion-transmitted infections to create inactivated tumor cells that can subsequently be used, in combination with Th1-promoting adjuvants, to stimulate an immune response 

 in preclinical models (5). This photochemical approach uses Riboflavin (vitamin B2) and UV light of specific wavelengths to generate specific modifications to tumor cell DNA that prevent replication of these cells (6). The specific nature of the chemistry for nucleic acid modification without alteration of proteins and antigens in the tumor cells yields cells that lack the ability to replicate but maintain their native antigen profile. In addition, these treated cells maintain cell metabolism and protein expression following treatment like blood cells treated similarly without the ability to replicate (7).

Tumor cells treated by this process were evaluated in immune-competent mouse models and shown to slow tumor growth, reduce metastatic disease, and stimulate immune cell responses to tumor tissue (8). In canine studies, preparations of tumor cells at doses as low as 1E+06 tumor cells per dose stimulate immune cytokine production and reduce tumor cell immunosuppressive cytokine expression. Doses of product sufficient to treat autochthonous tumors in dogs without inducing adverse acute reactions were reported previously (9).

The production of these materials does not require 

 expansion of the cells, thus reducing turnaround time and complexity of providing the therapy to patients. In a canine cancer trial conducted at the Flint Animal Cancer Center at Colorado State University, we tested the logistical practicality of using a biopsy or excised tumor tissue obtained at the Cancer Center, processing it at the PhotonPharma labs at the Research Innovation Center on campus, and returning the products to the canine subjects within one to seven days of their initial clinic visit. Production of the inactivated cells requires less than two hours using equipment and disposables that already received a Conformitè Europëenne Mark and are available in routine clinical use in blood centers globally (10).

Injection of the inactivated cells in an autologous fashion allows for the generation of immune response targeted to the patient’s specific tumor type and all tumor antigens that may be present in that patient’s tumor. The extensive experience of using treated blood products, which include nucleated leukocytes and lymphocytes from allogeneic donors, demonstrates the safety of the infusion of nucleated cells treated by this process. Riboflavin is a substance that has an extensive toxicology profile showing its safety after exposure to UV light in the process that is utilized here for creating inactivated tumor cells (11).

The low-cost profile of the equipment and disposables required for producing the inactivated cells via this method also has distinct advantages that may increase the accessibility of this therapy in patients with solid-organ tumors. As extensive 

 manipulation of the product is not required, production costs for such a therapy can also be limited and made more accessible for patients, healthcare providers, and national health services. Such an approach could make cellular therapy methods available to populations in many regions of the world today for which these procedures are currently inaccessible due to cost and complexity of implementation.

Our initial focus of work is the development of this therapy for treating patients with breast cancer. These tumors are generally accessible via biopsy or surgical excision. Breast cancer patients also experience high levels of metastatic relapse potential due to the nature of these tumors and the fact that chemotherapy or radiation therapy may allow for the survival of small populations of resistant cells. An autologous therapy approach as outlined in this article has the potential to reduce the likelihood of disease relapse in this patient population. Such an approach could also add another dimension to treating other solid organ tumors. This could include follow-up in such patients in a way that boosts the efficacy of more conventional treatments. The specificity of the therapy to the patient’s own tumor makes a response in each patient more relevant to their specific tumor composition, providing a lower likelihood of immunological escape. In the end, tumor material that is otherwise discarded can be applied to defeat the disease that led to its existence to begin with.

F. Locke and J. Lin, “Are CAR T-Cell Therapies Worth the Costs?” 

R. Goodrich, et al., “The Antiviral and Antibacterial Properties of Riboflavin and Light: Applications to Blood Safety and Transfusion Medicine,” in 

, E. Silva and A. Edwards, Eds. pp. 83-113. (RSC Publishing, Cambridge, UK, 2006).

Raymond P. Goodrich, PhD, is the founder of PhotonPharma and executive director of infectious disease research center and professor, department of microbiology, immunology, and pathology, for Colorado State University. 

Amanda Guth, DVM, PhD, is the chief scientific officer of PhotonPharma. 

 is the president and CEO of PhotonPharma. Terry Opgenorth, PhD, is the executive in residence life sciences for CSU Ventures. 

MD, PhD, is the president of the Global Coalition for Adaptive Research.


Volume 34, Number 12
December 2021
Pages: 

When referring to this article, please cite it as R. 

Formulating Autologous Therapies for Cancer

Autologous tumor cells engineered for immune system stimulation can target unique metabolic, genomic, and phenotypic characteristics of cancer cells.

Jennifer Markarian is manufacturing editor of BioPharm International.

The use of isolators for aseptic manufacturing has increased, driven by the effectiveness of the equipment for product safety and by the increasing number of products that must be processed aseptically (1). Isolators operate as closed systems and can be designed with attached gloves for manual intervention or as “gloveless” for fully automated systems. The draft revision to the European Union’s Annex 1 good manufacturing practices (GMP) for manufacturing sterile medicinal products (2) provides direction for designing and cleaning isolators (1). Evaluating the design of the isolator system in relation to the contamination control strategy is crucial, explained Richard Denk, a senior consultant for Aseptic Processing and Containment at SKAN (1).

Automation inside isolators further reduces human intervention. Comecer, an ATS company, displayed a containment isolator integrated with the company’s new automated SuperTrak Pharma platform for asynchronous conveyance at INTERPHEX 2021. The automated system offers flexibility with individually controlled shuttles, which increases throughput while while using a less complex and smaller footprint than traditional automation systems.

Although isolators have been commonly used in larger-scale or automated aseptic fill/finish, the technology is pushing into smaller-volume areas. Robotic automation is beneficial for small batches because it allows flexibility and more rapid changeover (3). Syntegon introduced the modular, small-batch Versynta FFP [Flexible Filling Platform] in 2021. It uses a four-axis robot inside the isolator to handle containers for filling aseptic or highly potent liquid products and can fill up to 3600 containers per hour (4). The isolator’s air flow is optimized so first air is supplied to the containers without obstruction.

Syntegon’s Versynta microBatch is an automated production cell in a gloveless isolator for smaller batches in the range of 120–500 containers per hour. This system, which is being developed in partnership with Vetter, is planned to be launched in 2022 (3).

Innovative isolator equipment is being developed and used in cell and gene therapy (CGT) manufacturing, as more companies get involved in the emerging area of advanced therapy medicinal products (ATMPs). While these small-volume processes started out primarily as manual processes in biosafety cabinets, new equipment offers the option of closed and automated processing. Biosafety cabinets have lower initial capital costs, but they present higher risk of microbial or cross-contamination and must be used in a Grade B cleanroom. Isolators, however, are closed systems that reduce contamination risk. Another advantage of using an isolator are that they can be used in a Grade C or D cleanroom, which requires less capital expenditure and lower energy use. The lower cleanroom levels require less personal protective equipment (PPE), which is more comfortable for operators as well as saving in costs and environmental impact of PPE disposal, explains Matt Pridden, standard product development manager at Extract Technology. Inquiries into isolator technology for ATMPs have been increasing, says Pridden, primarily because of the higher level of safety and the need to meet the requirements of Annex 1. Use of a Grade A isolator is known by regulators and the industry as the lowest level of risk to sterility, he adds.

Cellectis, for example, is a gene-editing company that recently opened a facility where it is performing clinical-stage manufacturing of allogeneic, universal chimeric antigen receptor T-cell (UCART) immunotherapies. In each the facility’s two manufacturing suites, a custom designed, four-chamber isolator encloses all parts of the UCART manufacturing process. Glove ports allow operators to perform manual process steps in the isolator. A separate isolator is used in the filling suite for automated fill/finish of the vials. Steve Doares, Cellectis’ senior vice-president of US manufacturing and the site head of the new Raleigh, NC manufacturing site, explained that the isolators were chosen instead of biosafety cabinets to maximize product quality assurance and maintain aseptic conditions throughout (5).

SKAN’s new Cellana isolator is designed for GMP manufacturing of ATMPs. A modular design allows different components to be integrated as needed for the specific process, and it can be integrated with semi-automatic or automated filling systems. The Cellana isolator uses technology already established in SKAN’s other isolators, such as the SKANfog decontamination technology and a catalytic convertor that completely decomposes hydrogen peroxide before the air is exhausted. The isolator uses a Grade A unidirectional airflow with high efficiency particulate arrestance filtration in both the air supply and the air return ducts, which allows it to handle viral vector and potent compounds (6).

In July 2021, powder-containment equipment specialist Dec Group acquired Extract Technology, which manufactures aseptic and containment work environments including CGT isolators (7). The company’s Modular Cell Therapy Isolator can be configured from chambers for transfer, centrifuge, and processing that form a Grade A environment, which is decontaminated using vapor-phase hydrogen peroxide. Patent-pending dockable incubators can be connected and disconnected as needed to allow multiple cell cultures to be processed. Another key aspect is the ability to integrate equipment specific to a client process, says Pridden. Extract Technology’s isolators have been used for several years to produce allogeneic stem cell transplant therapies. The systems can also be used for autologous cell therapies. The closed system allows open manipulation within a Grade A environment.

Comecer offers several isolators specifically designed for CGT. For example, a standalone isolator workstation is designed for applications that require cell manipulation and incubation under GMP conditions. The company makes a docking incubator system that can be connected to its CGT isolators using the Comecer Flexy interface. Modular components allow more flexibility to adapt and scale any manufacturing process. In June 2021, Comecer partnered with G-CON to provide its CGT closed aseptic systems (isolators and fillers) prequalified and installed inside G-CON’s prefabricated POD cleanrooms. (8).

ILC Dover extended its single-use, flexible containment isolator technology to aseptic isolators with the introduction of the soloPURE flexible aseptic isolator (9). The isolator’s closed, Grade-A aseptic environment can be used for aseptic processes and fill/finish tasks, including small-scale vial filling. The equipment has a small footprint, and the chamber has glove ports on both sides. The flexible chamber is gamma irradiated for use one time before disposal, or it can optionally be sterilized-in-place with vaporized hydrogen peroxide. The disposable chamber eliminates the need for cleaning and validation, which allows rapid changes from one product to another.

EC, Draft Revision to “Annex 1, Manufacture of Sterile Medicinal Products,” (Brussels, 2020).

Syntegon, “Versynta FFP,” Press Release, March 10, 2021.

J. Markarian, “Cellectis Opens GMP Manufacturing Facility,” 

SKAN, “Cellana Cell & Gene Therapy Isolators: Interview with Our Cell & Gene Task Force (3),” 

Dec Group, “Dec Group Announces Acquisition of Extract Technology,” Press Release, July 1, 2021.

Comecer, “Comecer Partners with G-CON to Speed Up the Implementation of Your Cell and Gene Therapy CGMP Manufacturing Facility,” Press Release, June 3, 2021.

ILC Dover, “ILC Dover Defines the Future of Sterile Manufacturing with the soloPURE Flexible Aseptic Isolator,” Press Release, April 13, 2021.

Jennifer Markarian is the manufacturing editor for 


Vol. 34, No. 12 
December 2021 
Pages: 26–27

When referring to this article, please cite it as J. Markarian, “Trends in Isolator Equipment,” 

Isolators move into smaller-volume processes, including cell and gene therapy manufacturing.

Trends in Isolator Equipment

For biological molecules to be safe and possess both high quality and efficacy, raw material quality control strategies are of the utmost importance. According to an article published by the Parenteral Drug Association (PDA), to achieve safety, quality, and efficacy, the following must be considered (1):

Supplier qualification system to assess raw material: origin risk, supply risk, and quality risk

Raw material quality testing strategies, including if specific tests are needed for particular raw materials.

Assessing supply risk, specifically, can include evaluating raw material availability, secondary options for supply, audits, and contractual agreements, while gauging quality risk may include evaluating consistency between lots, according to the PDA article (1). Raw material quality control strategies are essential to help ensure product quality and mitigate adventitious agent contamination risk (1).

Udo Losehand, head of global technical developers, Roquette—a supplier of biopharmaceutical raw materials—as well as Nate Fillipan, senior specialist, quality assurance; Stephanie Walsh, director, supply chain; and Paul Schildgen, director, manufacturing process optimization, all of Catalent Biologics—a manufacturer of biopharmaceutical raw materials—share with 

 how to determine the quality of raw materials, regulatory requirements to be aware of, supply chain challenges, and what factors must be considered when sourcing raw materials.

Behind the scenes: supplying raw materials

 What is the general procedure for making raw materials?

: Roquette is vertically integrated, and, therefore, performs every step of the manufacturing process in-house. This starts with the selection of appropriate grains and the extraction of starch, through to the manufacture of a high-quality product via enzymatic processing, hydrogenation, purification, packaging, and release testing.

 When a client approaches you for raw materials, what is that process like? Do you provide a consult as to whether specific materials are suitable for the client’s intended purpose?

: Indeed, for every customer request we receive, we evaluate the purpose and intended use of the ingredient in detail. This enables us to identify a product from our portfolio that is fit for purpose.

 What testing strategies do you have in place to ensure the quality of raw materials?

: We produce our raw materials according to good manufacturing practice (GMP) guidelines using processes validated for the manufacture of 

-compliant products. A combination of testing specifications for every batch, as well as routinely monitoring key performance indicators, means we can ensure the highest quality ingredients possible.

 How can the quality of biopharmaceutical raw materials be determined, specifically if it isn’t suitable for an intended application?

: Risk assessment and collaboration with the project sponsor [Catalent’s clients] are necessary for all instances where materials may not be suitable for an intended purpose. Specific use-testing, process development activities, and material qualification can be employed to help justify the use and mitigate risk.

 What does GMP mean for your company and its products?

: Excipients play a critical role in the manufacture of medicines, helping to preserve the efficacy, safety, and stability of active pharmaceutical ingredients (APIs). That’s why GMP manufacturing is such a significant part of Roquette Pharma Solutions’ DNA, to ensure each and every one of our customers’ products deliver its promised benefits to patients. Roquette has over 50 years of experience in the manufacture of pharmaceutical raw materials, complying with the appropriate GMP, for instance with ICH Q7 [International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use] for the APIs produced by Roquette (2). Meanwhile, our entire portfolio of biopharma raw materials meets GMP requirements for the International Pharmaceutical Excipients Council Federation’s validation of excipients. Our manufacturing site for biopharma raw materials is registered by the French Health Authority and audited regularly by the various regulatory agencies (e.g., FDA) as well.

 GMP can mean different things to different suppliers. How can a company ensure a supplier’s GMP standard meets its bioprocessing needs?

 From a supply chain perspective, providing our suppliers with clear expectations and regular feedback with regard to the GMP standard is critical to assuring that these suppliers will operate in a manner—and supply products—that will meet our needs. We do this at the site level with our material on-boarding process (including impact assessment), material specification process, and supplier complaint process. At the corporate level, expectation-setting and feedback are facilitated through supplier-relationship management (SRM) programs and the global supplier quality management audit process.

 When deciding where to source raw materials and what suppliers to work with, what factors must be considered? What could constitute an automatic pass for select raw materials?

 Biopharmaceutical manufacturing now includes single-use consumables, which make up the majority of raw materials. Single-use consumables and equipment technologies are interdependent, and identifying a supplier means looking at the capability, reliability, and compliance of both the equipment and supporting consumables.

: Generally, for other materials, the highest grade with the most appropriate compendial label claims is often the most desirable. Materials that are sold as research-grade are often much harder to justify because they may not have appropriate controls to prevent ingress of adventitious agents, may lack transparency to the original manufacturer, or are unsupported by change management, which would complicate their use.

 Normally, we also prefer to purchase directly from the manufacturer of a raw material or consumable. If we end up having to source from a distributor, transparency, with regard to the actual manufacturer, is paramount, as is the ability of the distributor (and manufacturer) to deploy rigorous change controls on the products that they distribute (or manufacture). A major factor in our decision to appoint a new supplier is our routine material impact assessment.

Supplying raw materials from a regulatory perspective

 What regulatory requirements do you have to be mindful of when supplying raw materials?

: Alongside GMP manufacturing and compliance with the standard monographs from the 

, Roquette ensures that additional requirements, specific to the needs of the biopharmaceutical manufacturing process, are met. This includes low endotoxin levels as well as the absence of relevant organic impurities, controlled bioburden, and more.

 Describe the difference between raw materials standards in the United States vs. globally.

: Typically, regulations for different regions are set by individual medical agencies. For example, the European Medicines Agency in the European Union or FDA in the US. Differences amongst such regulations are largely historical and stem from different manufacturing or quality standards. In recent years, thanks to the ICH program, significant steps have been initiated to standardize monographs as well as analytical methods (3).

 What supply chain concerns are present for sourcing raw materials?

: Roquette reduces the potential risk of a fragmented supply chain via its vertical integration, through which we can control every step of the manufacturing process of our biopharma raw materials in-house. In addition, close collaboration with our customers and a global distribution network, with the potential for local stock-keeping, further reduces the risk of a supply chain disruption.

 Critical biopharmaceutical raw materials are often proprietary and single-sourced. An effective sales and operations planning process drives appropriate mitigations to any potential supply-chain risks. Managing a reliable supply chain during extreme market volatility is also a critical concern. Keys to success in these challenging times are regular communication to raise awareness, transparency (both internally and externally), adaptive replenishment models, and operational flexibility.

 Effective and adaptive SRM programs, which include executive-level participation from both the supplier and the customer, where materials are single-sourced are also a means of mitigating potential supply chain challenges. Regular reviews and, if necessary, rapid adjustment to replenishment models are further mitigating actions. With regard to the need for flexibility, I would say that includes actively deploying backup sources of supply or working with what materials are available, versus those in constrained supply. Good internal communications, to raise awareness of situations, can further reduce risks. These should span all functional areas, with customer-facing functions, and even with customers themselves.

Constant and effective communication is necessary for every step of the process for sourcing biopharmaceutical raw materials, along with being aware of GMP guidelines as well as other guidelines (e.g., ICH) and regulatory expectations for different regions. While single-sourced biopharmaceutical raw materials can present supply chain challenges, adaptive replenishment models, operational flexibility, and general flexibility can help to mitigate these risks. But before all of this, it’s important for manufacturers and suppliers to carefully assess the capability, reliability, and compliance of both the equipment and supporting single-use consumables. As in most things in life, communication and upfront research are key.

A. Möritz, “Strategies for Controlling Raw Materials in Biologics Manufacturing,” PDA, 


Volume 34, Number 12
December 2021
Pages: 21–23

When referring to this article, please cite it as M. Rivers, "Ensuring Quality When Sourcing Biopharmaceutical Raw Materials," 

Defining expectations, such as GMPs, is paramount for sourcing the right biopharmaceutical raw materials.

Ensuring Quality When Sourcing Biopharmaceutical Raw Materials 

Feliza Mirasol is the science editor for 

Managing residual impurities in downstream processing is a challenge, even for traditional biologics such as monoclonal antibodies (mAbs). When it comes to managing residual impurities in downstream processing for emerging biotherapeutics, such as gene therapy or even cell therapy, those challenges are heightened due to the complex nature of the end product itself as well as the complicated bioprocesses needed to manufacture these types of products.

One such challenge in downstream bioprocessing is the requirement that the purification process be reliable and predictable to make product that is suitable for human use. It is thus imperative that during the downstream purification process, impurities such as host cell protein (HCP), DNA, adventitious and endogenous viruses, endotoxins, aggregates, and other species are removed while maintaining an acceptable product yield (1). Furthermore, impurities are also sometimes introduced during the purification process itself, and these must also be removed. Examples of impurities resulting from the processes themselves include leached Protein A, extractables from resins and filters, process buffers, and agents (e.g., detergents) that may have been used for viral clearance (1).

Recovery of an antibody product from mammalian cell culture typically starts with the cell harvest stage, where cells and cell debris are removed. The resulting clarified, filtered fluid is further purified in chromatography, which is a well-established process. In contrast, more complex emerging therapies, such as gene therapy products, present a unique set of challenges for controlling residual impurities. Because current gene therapy products are viral vector-based, this poses additional challenges to controlling the presence of HCPs. For gene therapies intended for severe indications that are placed on an accelerated development pathway, one critical challenge is the conflict between time-to-market pressures and the timelines required to develop a product-specific immunoassay (2).

As with mAbs, HCP content in viral-vector products is a critical quality attribute. HCP content is highly relevant to the safety of a gene therapy product (2). HCPs pose a risk directly through toxicity, hypersensitivity, endotoxin shock, or their own inherent biological activity. They may also pose a risk through immunogenicity, in which the HCPs themselves become the direct targets of the immune response. The presence of residual HCPs can also contain enzymes that can cause a gradual breakdown in protein components, thus compromising product stability (3).

Gene therapies that use viral vectors, such as adeno-associated virus (AAV) vectors, are challenging specifically because they contain viral material. Thus, removing impurities becomes especially complex when trying to distinguish between product and product-related impurities that closely resemble the vector itself. Thus there is a need to optimize both the upstream cell culture processes to reduce biosynthetic generation of these impurities and the downstream purification processes to reduce or remove these impurities without removing the desired product (4).

Appropriate and discerning analytical methods are key to managing residual impurities in emerging biotherapies. The ability to detect specific impurities in viral-vector-based gene therapies, for instance, allows for appropriate removal and process control over process-related impurities. However, the different analytical techniques available to the industry require a range of expertise in not only how to use them, but how to correctly interpret the data generated from these techniques.

The broad range of impurities that can show up in viral vector drug substance/drug product manufacturing needs comprehensive analysis. These analytical techniques include enzyme-linked immunosorbent assay (with specific antibodies), quantitative polymerase chain reaction (PCR) or digital-droplet PCR (ddPCR) assays using specific targeted amplicons, sodium dodecyl sulphate–polyacrylamide gel electrophoresisand capillary electrophoresis, high-performance liquid chromatography, analytical ultracentrifugation, fluorescence-activated cell sorting, and mass spectrometry (5).

Viral vectors themselves can be characterized with analytical techniques similarly used for traditional biotherapeutics; however, because understanding of viruses and virus functions remains incomplete, this fundamental lack of information complicates the issue of thoroughly characterizing viral vectors with certain analytical methods. Viral vectors consist of both protein and nucleic acid components. Methods must be used to characterize not only the viral capsid itself, but also the encapsulated genome (i.e., the “gene therapy”) (5). Complicating that is the challenge of then identifying and separating the product’s genetic signature from the genetic signature of residual impurities at the end of a manufacturing run, particularly impurities that closely resemble the product itself.

Confounding signals pose challenges to analytical methods necessary for managing residual impurity removal in biotherapeutic manufacturing.